Altered Coordination of Individual Catalytic Steps in Different and Evolved Inteins Reveals Kinetic Plasticity of the Protein Splicing Pathway

Protein splicing performed by inteins provides powerful opportunities to manipulate protein structure and function, however, detailed mechanistic knowledge of the multistep pathway to help engineering optimized inteins remains scarce. A typical intein has to coordinate three steps to maximize the product yield of ligated exteins. We have revealed a new type of coordination in the Ssp DnaB intein, in which the initial N–S acyl shift appears rate-limiting and acts as an up-regulation switch to dramatically accelerate the last step of succinimide formation, which is thus coupled to the first step. The structure–activity relationship at the N-terminal scissile bond was studied with atomic precision using a semisynthetic split intein. We show that the removal of the extein acyl group from the α-amino moiety of the intein’s first residue is strictly required and sufficient for the up-regulation switch. Even an acetyl group as the smallest possible extein moiety completely blocked the switch. Furthermore, we inv...